Diagnostic method

ABSTRACT

The present invention concerns a method for the detection or monitoring of cancer using a biological sample selected from blood, plasma, serum, saliva, urine from an individual, said method comprising:
         (a) obtaining DNA from the said biological sample;   (b) digesting the DNA sample with one or more methylation-sensitive restriction enzymes;   (c) quantifying or detecting a DNA sequence of interest after step (b), wherein the target sequence of interest contains at least two methylation-sensitive restriction enzyme recognition sites; and   (d) comparing the level of the DNA sequence from the individual to a normal standard, to detect, prognosticate or monitor cancer.

CROSS REFERENCE TO RELATED PATENT APPLICATION

This application claims the benefit of the filing date of U.S.provisional patent application 60/847,499, filed Sep. 27, 2006, thedisclosure of which is herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to methods for diagnosis, prognosis ormonitoring of cancer in an individual, in particular using thedifferential methylation patterns in genes associated with cancers.

BACKGROUND TO THE INVENTION

It is well known that many tumor suppressor genes are methylated intumor cells. As such, the use of methylation markers has been suggestedfor the detection or monitoring of cancer in patients. A number ofdifferent methods have been proposed for detection of these methylatedsequences.

Methylation specific PCR (MSP) is the most commonly used method fordetecting methylated or unmethylated DNA. MSP involves the step ofbisulfite conversion. Sodium bisulfite is used to deaminate cytosine touracil while leaving 5-methyl-cytosine intact. Methylation-specific PCRuses PCR primers targeting the bisulfite induced sequence changes tospecifically amplify either methylated or unmethylated alleles.Bisulfite conversion destroys about 95% of the DNA. Since DNAconcentrations are typically very low in the serum or plasma, a 95%reduction in DNA results in a detection rate of less than 50%.

Alternative methods use restriction enzymes that digest specificallyeither the methylated or unmethylated DNA. Enzymes that cut specificallymethylated DNA are rare. However, enzymes that cut specificallyunmethylated DNA are more readily available. Detection methods thenestablish whether digestion has occurred or not, and thus depending onthe specificity of the enzyme used, allows detection of whether theunderlying DNA was methylated or unmethylated and thus associated withcancer or not.

Methylation-sensitive enzyme digestion has been previously proposed. Forexample, Silva et al, British Journal of Cancer, 80:1262-1264, 1999conducted methylation-sensitive enzyme digestion followed by PCR.However, as noted by the authors Yegnasubramanian et al, Nucleic AcidsResearch, Vol. 34, No. 3, 2006 e19, such methods are plagued by thenumber of false-positives that are generated.

The present invention seeks to provide enhanced methods ofmethylation-sensitive detection which eliminate or reduce falsepositives and/or false negatives.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided a method forthe detection or monitoring of cancer using a biological sample selectedfrom blood, plasma, serum, saliva, urine from an individual, said methodcomprising:

-   -   (a) obtaining DNA from the said biological sample;    -   (b) digesting the DNA sample with one or more        methylation-sensitive restriction enzymes;    -   (c) quantifying or detecting a DNA sequence of interest after        step (b), wherein the target sequence of interest contains at        least two methylation-sensitive restriction enzyme recognition        sites; and    -   (d) comparing the level of the DNA sequence from the individual        to a normal standard, to detect, prognosticate or monitor        cancer.

In a preferred aspect of the present invention, the polymerase chainreaction is used in step (c). Preferably, the methylation-sensitiverestriction enzyme recognises DNA sequences which have not beenmethylated. The target sequence is a sequence susceptible to methylationin cancer patients so that an unmethylated target sequence in a normalpatient is digested and is not amplified by the polymerase chainreaction, whereas in a cancer patient, the target sequence is methylatedand is not digested by the enzyme and can subsequently be quantified ordetected, for example using the polymerase chain reaction.

The methods of the present invention can be used to predict thesusceptibility to cancer of the individual, to assess the stage ofcancer in the individual, to predict the likelihood of overall survivalfor the individual, to predict the likelihood of recurrence for theindividual or to assess the effectiveness of treatment in theindividual.

In accordance with another aspect of the present invention, there isprovided a method for the detection or monitoring of cancer using abiological sample selected from blood, plasma, serum, saliva, urine froman individual, said method comprising:

-   -   (a) obtaining DNA from the said biological sample;    -   (b) digesting the DNA sample with one or more        methylation-sensitive restriction enzymes;    -   (c) quantifying or detecting a DNA sequence of interest after        step (b) wherein the DNA sequence is a sequence comprising part        or all of RASSF1A; and    -   (d) comparing the level of the DNA sequence from the individual        to a normal standard, to detect, prognosticate or monitor        cancer.

In accordance with a further aspect of the invention, there is providedprobes, primers and kits for use in the method of the invention. Inparticular, there is provided:

a detectably-labelled probe for the detection or monitoring of cancer ina biological sample selected from blood, plasma, serum, saliva, urinefrom an individual, which comprises the sequence shown in SEQ ID NO: 4;

a set of primers for the detection or monitoring of cancer in abiological sample selected from blood, plasma, serum, saliva, urine froman individual, which comprises a primer comprising the sequence shown inSEQ ID NO: 2 and a primer comprising the sequence shown in SEQ ID NO: 3;

a kit for the detection or monitoring of cancer in a biological sampleselected from blood, plasma, serum, saliva, urine from an individual,which comprises the probe of the invention and the set of primers of theinvention

a detectably-labelled probe for use as a control during the detection ormonitoring of cancer in a biological sample selected from blood, plasma,serum, saliva, urine from an individual, which comprises the sequenceshown in SEQ ID NO: 7;

a set of primers for use as a control during the detection or monitoringof cancer in a biological sample selected from blood, plasma, serum,saliva, urine from an individual, which comprises a primer comprisingthe sequence shown in SEQ NO: 5 and a primer comprising the sequenceshown in SEQ ID NO: 6; and

a kit for use as a control during the detection or monitoring of cancerin a biological sample selected from blood, plasma, serum, saliva, urinefrom an individual, which comprises the control probe of the inventionand the set of control primers of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1: concentration of methylated RASSF1A in patients' plasma.

FIG. 2: changes of plasma methylated RASSF1A levels after surgicalresection of hepatocellular carcinoma (HCC).

FIG. 3: methylated RASSF1A sequence concentration in plasma prior tosurgical resection is predictive of patient survival after surgicalresection.

FIG. 4: methylated RASSF1A sequence concentration in plasmapost-surgical resection is predictive of patients survival aftersurgical resection.

FIG. 5: concentration of methylated RASSF1A detected in the plasma ofnasopharangeal carcinoma (NPC) patients correlated with Epstein-Barrvirus (EBV) DNA concentration.

FIG. 6: Genomic sequence of the promoter and the first exon of theRASSF1A gene (SEQ ID NO: 1). The recognition sequence of themethylation-sensitive restriction enzyme BstUI is underlined and thetarget sequence for PCR detection is highlighted in bold. There are 5BstUI enzyme restriction sites in the target sequence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method to assess, diagnose,prognosticate or monitor the presence or progression of tumors in anindividual. The method involves the use of a methylation-sensitiverestriction enzyme to digest DNA sequences. DNA sequences of interestare selected which contain at least two restriction sites which may ormay not be methylated. The method is preferably carried out withmethylation-sensitive restriction enzymes which preferentially cleaveuninethylated sequences compared to methylated sequences. Methylatedsequences remain undigested and are detected. Digestion of unmethylatedsequences at at least one of the methylation-sensitive restrictionenzyme sites results in the target sequence not being detected oramplifiable. Thus a methylated sequence can be distinguished from anunmethylated sequence. In one embodiment of the invention, the quantityof uncut target sequence detected in a biological sample, e.g. plasma orserum of cancer patients is higher than that demonstrated in abiological sample of the same type of healthy or cancer-free individualssince the target sequences are more highly methylated in cancer patientsthan healthy individuals.

In the alternative, restriction enzymes which out methylated DNA can beused. Unmethylated DNA sequences are not digested and can be detected.In another embodiment of this invention, lower quantities of the uncutDNA sequence are detected in a biological sample, e.g. plasma, or serumof cancer patients when compared with that demonstrated in a biologicalsample of the same type in cancer-free individuals.

In a preferred embodiment according to the present invention, the targetsequence is detected by amplification by PCR. Real-time quantitative PCRcan be used. Primer sequences are selected such that at least twomethylation-sensitive restriction enzyme sites are present in thesequence to be amplified using such primers. The methods in accordancewith the present invention do not use sodium bisulfite. Amplification bya suitable method, such as PCR, is used to detect uncut target sequence,and thus to identify the presence of methylated DNA which has not beencut by restriction enzymes.

In accordance with the present invention, any suitablemethylation-sensitive restriction enzyme can be used. Examples ofmethylation-sensitive restriction enzymes that cut unmethylated DNA arelisted in Table I below.

TABLE I Examples of methylation-sensitive restriction enzymes:Effect of CpG Recognition methylation on Enzyme sequenceenzyme restriction* AatII GACGTC blocked AftI CACGTC blockedBstUI, Bsh1236I CGCG blocked Bsh1285I CGRYCG blocked BshTI ACCGGTblocked Bsp68I TCGCGA blocked Bsp119I TTCGAA blocked Bsp143II RGCGCYblocked Bsu15I ATCGAT blocked CseI GACGC blocked Cff10I RCCGGY blockedCfr42I CCGCGG blocked CpoI CGGWCCG blocked Eco47III AGCGCT blockedEco52I CGGCCG blocked Eco72I CACGTG blocked Eco105I TACGTA blocked EheIGGCGCC blocked Esp3I CGTCTC blocked FspAI RTGCGCAY blocked HhaI; Hin6IGCGC blocked Hin1I GRCGYC blocked HpaII CCGG blocked Kpn2I TCCGGAblocked MluI ACGCGT blocked NotI GCGGCCGC blocked NsbI TGCGCA blockedPauI GCGCGC blocked PdiI GCCGGC blocked Pfi23II CGTACG blocked Ppu21IYACGTR blocked Psp1406I AACGTT blocked PvuI CGATCG blocked SalI GTCGACblocked SgsI GGCGCGCC blocked SmaI CCCGGG blocked SmuI CCCGC blockedSsiI CCGC blocked. TaiI ACGT blocked TauI GCSGC blocked The letter codesin the recognition sequences represent different combinations ofnucleotides and are summarised as follows: R = G or A; Y = C or T; W = Aor T; M = A or C; K = G or T; S = C or G; H = A, C or T; V = A, C or G;B = C, G or T; D = A, G or T; N = G, A, T or C. The CpG dinucleotide(s)in each recognition sequence is/are underlined. The cytosine residues ofthese CpG dinucleotides are subjected to methylation. *The methylationof the cytosine of the CpG dinucleotides in the recognition sequencewould prevent enzyme cutting of the target sequence.

The target sequence includes two or more methylation-sensitiverestriction enzyme sites. Such sites may be recognised by the same ordifferent enzymes. However, the sites are selected so that at least twosites in each sequence are digested when unmethylated when using enzymeswhich preferentially cleave unmethylated sequences compared tomethylated sequences.

In a less preferred embodiment the target sequence contains at least twosites which are cut or cleaved by restriction enzymes whichpreferentially cleave methylated sequences. The two or more sites may becleaved by the same or different enzymes.

Any suitable DNA methylation marker may be used in accordance with thepresent invention. Such DNA methylation markers are those where theselected sequence shows a different methylation pattern in cancerpatients compared to normal individuals. Suitable markers are selectedsuch that the sequence to be amplified contains at least twomethylation-sensitive restriction enzyme sites. Generally suchmethylation markers are genes where promoter and/or encoding sequencesare methylated in cancer patients. Preferably the selected sequences arenot methylated or are methylated to a lesser extent in non-cancer orcancer-free individuals.

Suitable DNA methylation markers include, for example, RASSF1A. Indeed,RASSF1A has proved to be particularly effective for use in detection ormonitoring of cancer in an individual. Thus, in accordance with analternative aspect of the present invention, there is provided a methodfor the detection or monitoring of cancer using a biological sampleselected from blood, plasma, serum, saliva, urine from an individual,said method comprising:

(a) obtaining DNA from the said biological sample;

(b) digesting the DNA sample with one or more methylation-sensitiverestriction enzymes;

(c) quantifying or detecting a DNA sequence of interest after step (b)wherein the DNA sequence is a sequence comprising part or all ofRASSF1IA; and

(d) comparing the level of the DNA sequence from the individual to anormal standard, to detect, prognosticate or monitor cancer.

The tumor types associated with RASSF1A promoter hypermethylation arelisted in Table II below.

TABLE II Frequencies of RASSF1A promoter hypermethylation in differenttypes of cancers (ordered in descending frequency): Frequencies ofRASSF1A promoter Cancer types hypermethylation References Liver 93%-100%  (1-4) Breast 49%-95%  (5-8) Lung (small cell) 79%-88% (9, 10)Prostate 71%-83% (11-13) Melanoma 41%-75% (14, 15) Pancreas 64% (16)Kidney 36%-64% (17-19) Bladder 47%-60% (20, 21) Colon 12%-45% (19,22-24) Ovary 30%-40% (19, 25, 26) Lung (non-small cell) 28%-40% (9, 27,28)

REFERENCES FOR TABLE II

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Epigenetic inactivation of RASSF1A in lung and breast    cancers and malignant phenotype suppression. J Natl Cancer Inst    2001; 93:691-9.-   6. Mehrotra I, Vali M, McVeigh M, Kominsky S L, Fackler M J,    Lahti-Domenici J, et al. Very high frequency of hypermethylated    genes in breast cancer metastasis to the bone, brain, and lung. Clin    Cancer Res 2004; 10:3104-9.-   7. Fackler M J, McVeigh M, Evron E, Garrett E, Mehrotra J, Polyak K,    et al. DNA methylation of RASSF1A, HIN-1, RAR-beta, Cyclin D2 and    Twist in in situ and invasive lobular breast carcinoma. Int J Cancer    2003; 107:970-5.-   8. Yeo W, Wong W L, Wong N, Law B K, Tse G M, Zhong S. High    frequency of promoter hypermethylation of RASSF1A in tumorous and    non-tumourous tissue of breast cancer. Pathology 2005; 37:125-30.-   9. Grote H J, Schmiemann V, Geddert H, Booking A, Kappes R, Gabbert    H E, et al. Methylation of RAS association domain family protein 1A    as a biomarker of lung cancer. 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Marini A, Mirmohammadsadegh A, Nambiar S, Gustrau A, Ruzicka T,    Hengge U R. Epigenetic inactivation of tumor suppressor genes in    serum of patients with cutaneous melanoma. J Invest Dermatol 2006;    126:422-31.-   16. Dammann R, Schagdarsurengin U, Liu L, Otto N, Gimm O, Dralle H,    et al. Frequent RASSF1A promoter hypermethylation and K-ras    mutations in pancreatic carcinoma. Oncogene 2003; 22:3806-12,-   17. Tokinaga K, Okuda H, Nomura A, Ashida S. Furibata M, Shuin T.    Hypermethylation of the RASSF1A tumor suppressor gene in Japanese    clear cell renal cell carcinoma. Oncol Rep 2004; 12:805-10.-   18. Dulaimi E, Ibanez de Caceres I, Uzzo R G, Al-Saleern T,    Greenberg R E, Polascik T J, et al, Promoter hypermethylation    profile of kidney cancer. Clin Cancer Res 2004; 10:3972-9.-   19. Yoon J H, Dammann R, Pfeifer G P. Hypermethylation of the CpG    island of the RASSF1A gene in ovarian and renal cell carcinomas. Int    J Cancer 2001; 94:212-7.-   20. 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Other preferred tumor suppressor genes showing hypermethylation intumors are listed in Table III.

TABLE III Examples of tumor suppressor genes which promoter regions arefrequently inactivated by methylation in cancers: Types of cancer inwhich the gene may be aberrantly Tumor suppressor gene methylatedReferences APC colorectal, breast, head and  (1-13) neck, esophagus,bladder, prostate, stomach, lung, kidney DAP-kinase pancreas, stomach,lung, (9, 10, 14-19) colorectal, breast, cervix, nasopharynx E-cadherinbreast, lung, stomach, (8, 20-27) colorectal, prostate, bladder, cervix,kidney GSTPI lung, stomach, bladder, (8, 9, 11, 12, 28-31) prostate,breast, cervix hMLH1 stomach, colorectal, cervix, (6, 9, 16, 21, 31-34)liver, esophagus, lung, ovary, prostate MGMT lung, colorectal, bladder,(8, 11, 13, 15-17, 28, cervix, breast, esophagus, 31, 35, 36) prostate,nasopharynx, kidney NORE1A kidney, lung, breast, colon (37-39) p14colorectal, bladder, (11, 13, 16, 19, 40-42) nasopharynx, kidney,stomach, breast p15 bladder, nasopharynx, kidney, (7, 11, 19, 27, 43-46)multiple myeloma, colorectal, lung, ovary, stomach P16INK4a lung,stomach, bladder, cervix, (8, 9, 11, 15, 17, 18, nasopharynx, breast,prostate, 27, 28, 35, 43, 44, 47-49) kidney, liver, colorectal,pancreas, leukemia, multiple myeloma, thyroid RARbeta lung, breast,nasopharynx, (8, 12, 13, 19, 23, 30, prostate, kidney, stomach 35, 50)SOCS1 colorectal, leukemia, stomach, (51-57) ovary, liver, pancreas Rbretinoblastoma, lung, (58-61) esophagus, stomach VHL kidney (13, 62)

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In accordance with the method of the present invention a sample is takenor obtained from the patient. Suitable samples include blood, plasma,serum, saliva and urine. Samples to be used in accordance with thepresent invention include whole blood, plasma or serum. Methods forpreparing serum or plasma from whole blood are well known among those ofskill in the art. For example, blood can be placed in a tube containingEDTA or a specialised commercial product such as Vacutainer SST (BectonDickenson, Franklin Lake, N.J.) to prevent blood clotting, and plasmacan then be obtained from whole blood through centrifugation. Serum maybe obtained with or without centrifugation following blood clotting. Ifcentrifugation is used then it is typically, though not exclusivelyconducted at an appropriate speed, for example, 1500-3000×g. Plasma orserum may be subjected to additional centrifugation steps before beingtransferred to a fresh tube for DNA extraction.

Preferably, DNA is extracted from the sample using a suitable DNAextraction technique. Extraction of DNA is a matter of routine for oneof skill in the art. There are numerous known methods for extracting DNAfrom a biological sample including blood. General methods of DNApreparation, for example described by Sambrook and Russell, MolecularCloning a Laboratory Manual, 3^(rd) Edition (2001) can be followed.Various commercially available reagents or kits may also be used toobtain DNA from a blood sample.

In accordance with the invention, the DNA containing sample is incubatedwith one or more restriction enzyme(s) which preferentially cutunmethylated DNA under conditions such that where two or morerestriction enzyme sites are present in the target sequence in theunmethylated state, the restriction enzyme(s) can cut the targetsequence at at least one such site. In accordance with an alternativeaspect of the invention, a DNA sample is incubated with one or morerestriction enzymes which only cut methylated DNA under conditions suchthat where two or more restriction enzyme sites are present in themethylated state, the restriction enzyme(s) can cut the target sequenceat at least one such site.

Preferably samples are incubated under conditions to allow completedigestion. This may be achieved, for example by increasing theincubation times and/or increasing the quantity of the enzyme used.Typically, the sample will be incubated with 100 active units ofmethylation-sensitive restriction enzyme for a period of up to 16 hours.It is a matter of routine for one of skill in the art to establishsuitable conditions based on the quantity of enzyme used.

After incubation, uncut target sequences are detected. Preferably, thesesequences are detected by amplification, for example using thepolymerase chain reaction (PCR).

DNA primers are designed to amplify a sequence containing at least twomethylation-sensitive restriction enzyme sites. Such sequences can beidentified by looking at DNA methylation markers and identifyingrestriction enzyme sites within those markets which are recognised bymethylation-sensitive enzymes. For example using the recognitionsequences for the methylation-sensitive enzymes identified in Table I,suitable target sequences can be identified in the methylation markerslisted in Table III.

Using RASSF1A as an example, the target sequence may comprise part orall of the promoter sequence and/or exon 1 of the RASSF1A gene. Thesequence for the promoter and exon 1 is set out in FIG. 6 (SEQ NO: 1).In a preferred embodiment the target sequence for detection is thathighlighted in bold in FIG. 6 (residues 1142 to 1269 of SEQ ID NO: 1).In a more preferred embodiment residues 1142 to 1269 of SEQ ID NO: 1 areamplified using (a) a primer comprising or consisting of the sequenceshown in SEQ ID NO: 2 and (b) a primer comprising or consisting of thesequence shown in SEQ ID NO: 3. In another more preferred embodimentresidues 1142 to 1269 of SEQ ID NO: 1 are detected using adetectably-labelled probe comprising the sequence shown in SEQ ID NO: 4.In an even more preferred embodiment residues 1142 to 1269 of SEQ ID NO:1 are detected using a detectably-labelled probe comprising the sequenceshown in SEQ ID NO: 4 and no additional nucleotides.

When using methylation-sensitive enzymes, altered quantities of thetarget sequence will be detected depending on the methylation status ofthe target sequence in a particular individual. In the preferred aspectof the present invention using methylation-sensitive restriction enzymeswhich preferentially cut unmethylated DNA, the target sequence will notbe detected in the unmethylated state, for example in a healthyindividual. However, where the target sequence is methylated, forexample in a selected sample from a cancer patient, the target sequenceis not cut by the restriction enzyme and the target sequence can thus bedetected by PCR.

Thus, the method can be used to determine the methylation status of thetarget sequence and provide an indication of the cancer status of theindividual.

The methods of the present invention may additionally includequantifying or detecting a control sequence. The control sequence isselected which does not show aberrant methylation patterns in cancer. Inaccordance with a preferred aspect of the present invention, the controlsequence is selected to contain at least two methylation-sensitiverestriction enzyme recognition sites. Preferably, the control sequenceis selected to contain the same number of methylation-sensitiverestriction enzyme recognition sites as the DNA sequence of interest.Typically the presence or absence of such control sequences is detectedby amplification by the polymerase chain reaction after digestion withthe methylation-sensitive restriction enzyme(s). Such control sequencescan be used to assess the extent of digestion with the one or moremethylation-sensitive restriction enzymes. For example, if afterdigestion with the methylation-sensitive restriction enzyme(s) controlsequences are detectable, this would indicate that the digestion is notcomplete and the methods can be repeated to ensure that completedigestion has occurred. Preferably the control sequence is selected tocontain the same methylation-sensitive restriction enzyme sites that arepresent in the target sequence. In a preferred embodiment the controlsequence is β-actin. In a more preferred embodiment a target sequence inthe β-actin is amplified using (a) a primer comprising or consisting ofthe sequence shown in SEQ ID NO: 5 and (b) a primer comprising orconsisting of the sequence shown in SEQ ID NO: 6. In an even morepreferred embodiment the target sequence in β-actin (which is amplifiedusing primers comprising SEQ NOs: 5 and 6) is detected using adetectably-labelled probe comprising the sequence shown in SEQ ID NO: 7.In an even more preferred embodiment the target sequence is detectedusing a detectably-labelled probe comprising the sequence shown in SEQII) NO: 7 and no additional nucleotides.

The present methods can be used to assess the tumor status of anindividual. The methods can be used, for example, in the diagnosisand/or prognosis of cancer. The methods can also be used to monitor theprogress of cancer, for example, during treatment. The methods can alsobe used to monitor changes in the levels of methylation over time, forexample to assess the susceptibility of an individual to cancer, and theprogression of the disease. The methods can also be used to predict theoutcome of disease or the likelihood of success of treatment

In a preferred aspect of the present invention the target sequence isRASSF1A and is used in the diagnosis of cancer. For example, RASSF1Amethylation can be used to detect and monitor hepatocellular ornasopharyngeal carcinoma. The method is particularly useful inmonitoring the susceptibility of a hepatitis B carrier or a hepatitis Ccarrier to hepatocellular carcinoma.

In another aspect of the invention, there is provided probes and primersfor use in the method of the invention. Firstly, there is provided a setof primers or a detectably-labelled probe for the detection ormonitoring of cancer in a biological sample selected from blood, plasma,serum, saliva, urine from an individual. The set of primers comprises orconsists of (a) a primer comprising or consisting of the sequence shownin SEQ ID NO: 2 and (b) a primer comprising or consisting of thesequence shown in SEQ ID NO: 3. The set of primers is capable ofamplifying residues 1142 to 1269 of SEQ ID NO: 1 (i.e. part of thepromoter and first exon of the RASSF1A gene). The detectably-labelledprobe comprises the sequence shown in SEQ ID NO: 4 and is capable ofdetecting residues 1142 to 1269 of SEQ ID NO: 1. The detectably-labelledprobe preferably comprises the sequence shown in SEQ ID NO: 4 and noadditional nucleotides. The detectably-labelled probe is most preferablythe probe used in the Examples.

Secondly, there is provided a set of primers and a detectably-labelledprobe for use as a control during the detection or monitoring of cancerin a biological sample selected from blood, plasma, semi, saliva, urinefrom an individual. The set of primers comprises or consists of (a) aprimer comprising or consisting of the sequence shown in SEQ ID NO: 5and (b) a primer comprising or consisting of the sequence shown in SEQ110 NO: 6. The set of primers is capable of amplifying a target sequencein β-actin. The detectably-labelled probe comprises the sequence shownin SEQ ID NO: 7 and is capable of detecting the target sequence inβ-actin that is amplified by the printers comprising SEQ ID NOs: 5 and6. The detectably-labelled probe preferably comprises the sequence shownin SEQ ID NO: 7 and no additional nucleotides. The detectably-labelledcontrol probe is most preferably the control probe used in the Examples.

The probes are detectably-labelled. The detectable label allows thepresence or absence of the hybridization product formed by specifichybridization between the probe and the target sequence to bedetermined. Any label can be used. Suitable labels include, but are notlimited to, fluorescent molecules, radioisotopes, e.g. ¹²⁵I, ²⁵S,enzymes, antibodies and linkers such as biotin.

In another aspect, there is provided kits for use in the method ofinvention. Firstly, there is provided a kit for the detection ormonitoring of cancer in a biological sample selected from blood, plasma,serum, saliva, trine from an individual. The kit comprises (a) a primercomprising or consisting of the sequence shown in SEQ NO: 2, (b) aprimer comprising or consisting of the sequence shown in SEQ ID NO: 3and (c) a detectably-labelled probe comprising the sequence shown in SEQID NO: 4 and optionally no additional nucleotides. The kit is capable ofamplifying and detecting residues 1142 to 1269 of SEQ ID NO: 1.

Secondly, there is provided a kit for use as a control during thedetection or monitoring of cancer in a biological sample selected fromblood, plasma, serum, saliva, urine from an individual. The kitcomprises (a) a primer comprising or consisting of the sequence shown inSEQ ID NO: 5, (b) a primer comprising or consisting of the sequenceshown in SEQ 1D NO: 6 and (c) a detectably-labelled probe comprising thesequence shown in SEQ ID NO: 7 and optionally no additional nucleotides.The kit is capable of amplifying and detecting a target sequence inβ-actin.

The kits of the invention may additionally comprise one or more otherreagents or instruments which enable the method of the invention asdescribed above to be carried out. Such reagents or instruments includeone or more of the following: suitable buffer(s) (aqueous solutions),PCR reagents, fluorescent markers and/or reagents, means to obtain asample from individual subject (such as a vessel or an instrumentcomprising a needle) or a support comprising wells on which reactionscan be done. Reagants may be present in the kit in a dry state such thatthe fluid sample resuspends the reagents. The kit may, optionally,comprise instructions to enable the kit to be used in the method of theinvention.

The invention is hereinafter described in more detail by reference tothe Examples below.

EXAMPLES

Sixty-three hepatocellular carcinoma (HCC) patients, as well as twogroups of controls, were recruited. The first control group consisted of15 healthy control subjects without chronic hepatitis B infection. Thesecond control group consisted of chronic hepatitis B carriers. For eachHCC patient, one sex-and age-matched chronic hepatitis B carrier wasrecruited as a control. Chronic hepatitis B carriers had increased riskof developing HCC and, thus, would be the target group for HCCscreening. Four milliliters of venous blood were collected from eachstudy subject into EDTA-containing tubes. Blood samples were centrifugedat 1,600 g for 10 min and the supernatant was re-centrifuged at 16,000 gfor 10 min. DNA was then extracted from 800 μL of plasma using theQIAamp mini kit (Qiagen, Hilden, Germany) and eluted with 50 μL of H₂O.Thirty-five microliters of plasma DNA were digested with 100 U of BstU Ienzyme, in 1× digestion buffer at 60° C. for 16 hours.

The concentration of plasma RASSF1A sequence was determined by real-timePCR using the primers 5′AGCCTGAGCTCATTGAGCTG3′ (SEQ ID NO: 2) and5′ACCAGCTGCCGTGTGG3′ (SEQ ID NO: 3), and the probe5′FAM-CCAACGCGCTOCGCAT(MGB)3′ (SEQ ID NO: 4). Each reaction contains 1×TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City), 300nM each of the primers and 85 nM of probes. 7.15 microliters ofenzyme-digested plasma DNA mixture (equivalent to 5 μL of undigestedplasma DNA) were used as the template for each PCR reaction. The thermalprofile was 50° C. for 2 minutes, 95° C. for 10 minutes, 50 cycles of95° C. for 15 seconds and 60° C. for 1 minute. All reactions were run induplicate and the mean quantity was taken. The RASSF1A amplicon embraced5 restriction sites of BstUI.

To ensure the completeness of the restriction enzyme digestion,real-time PCR targeting the β-actin gene was performed for each enzymedigested samples using the primers 5′ GCGCCGTTCCGAAAGTT3′ (SEQ ID NO: 5)and 5′CGGCGGATCGGCAAA3′ (SEQ ID NO: 6), and the probe5′FAM-ACCGCCGAGACCGCGTC(MGB)3′ (SEQ ID NO: 7). By bisulfite sequencing,the β-actin gene promoter was shown to be completely unmethylated inblood cells and HCC tissues. The β-actin amplicon is of similar size ofthe RASSF1A amplicon and contains identical number of BstUI enzymerestriction sites.

To investigate if the enzyme digestion efficiencies for unmethylatedRASSF1A and β-actin sequences were similar, aliquots of 1 μg of buffycoat were digested with 100 U of BstU I enzyme for different timeintervals (15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes,120 minutes, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours and 16 hours).The concentrations of RASSF1A and β-actin sequences were measured ineach sample after the enzyme digestion. As shown in FIG. 1, theconcentrations of RASSF1A and -actin sequences in these buffy coat DNAshowed a positive correlation (r=0.986, P<0.0001, Pearson correlation).As the enzyme digestion efficiencies for unmethylated RASSF1A andβ-actin sequences are similar, the completeness of the digestion ofunmethylated RASSF1A sequence should be reflected by the absence ofβ-actin sequence in the digested plasma DNA sample. Therefore, allsamples with positive β-actin signal were subject to further enzymedigestion until no β-actin sequence was detectable in the digestedplasma DNA sample.

After BstUI enzyme digestion, RASSF1A sequences were detected in theplasma of 59 (93%) of the 63 HCC patients and 37 (58%) of the 63 matchedchronic hepatitis B carriers (HBV carriers). These results are shown inFIG. 2. The median plasma RASSF1A concentrations of the HCC patients andchronic hepatitis B carriers were 770 copies/mL and 118 copies/mL,respectively. In contrast, RASSF1A was not detectable in the plasma ofany of the 15 healthy subjects.

FIG. 3 demonstrates that the survival probabilities of HCC patients withpreoperative plasma RASSF1A concentration of less than 550 copies/mLwere better than those with levels higher than 550 copies/mL (p=0,0359,Kaplan-Meier survival analysis).

Blood samples were collected from the HCC patients at 1 month after thesurgical resection of the tumor. In the 59 patients with detectableplasma RASSF1A before tumor resection, 45 of them (76%) showed areduction in the concentration after the operation. These results areshown in FIG. 4. Among these 45 patients, 13 of them had undetectableRASSF1A after the resection of tumor. The median RASSF1A concentrationdropped from 770 copies/mL to 250 copies/mL (p<0.0001, Wilcoxon test).

To further investigate if the quantitative analysis of plasma RASSF1Aafter methylation-sensitive restriction enzyme digestion is a genericmarker for cancers with aberrant methylation of RASSF1A, 67nasopharyngeal carcinoma (NPC) patients were recruited for the study ofthe correlation of the plasma concentrations ofenzyme-digestion-resistant RASSF1A and Epstein-Barr virus (EBV) DNA.Plasma EBV DNA is an established marker for NPC and has been shown toreflect tumor load (Lo Y M D, Chan L Y, Lo K W, Leung S F, Zhang J, ChanA T, Lee J C, Hjelm N M, Johnson P J, Huang D P. Quantitative analysisof cell-free Epstein-Barr virus DNA in plasma of patients withnasopharyngeal carcinoma. Cancer Res 1999; 59:1188-91). The results areshown in FIG. 5. EBV DNA and enzyme-digestion-resistant RASSF1A sequencewere detectable in the plasma of 65 (94%) and 37 (54%) patients,respectively. In the patients with detectable enzyme-digestion-resistantRASSF1A and EBV DNA, the plasma concentrations of the two DNA sequencesshowed a positive correlation (r=0.343, p=0.037, Spearman correlation).

1. A method for the detection or monitoring of cancer using a biologicalsample selected from blood, plasma, serum, saliva, urine from anindividual, said method comprising: (a) obtaining DNA from the saidbiological sample; (b) digesting the DNA sample with one or moremethylation-sensitive restriction enzymes; (c) quantifying or detectinga DNA sequence of interest after step (b), wherein the target sequenceof interest contains at least two methylation-sensitive restrictionenzyme recognition sites; and (d) comparing the level of the DNAsequence from the individual to a normal standard, to detect,prognosticate or monitor cancer.
 2. The method according to claim 1,wherein the polymerase chain reaction (PCR) is used in step (c), and thePCR amplicon contains at least two recognition sites for themethylation-sensitive restriction enzyme used in step (b).
 3. The methodaccording to claim 1, wherein real-time quantitative polymerase chainreaction (Q-PCR) is used in step (c) and the PCR amplicon contains atleast two recognition sites for the methylation-sensitive restrictionenzyme used in step (b).
 4. The method according to claim 1, wherein theDNA sequence quantified in step (c) is a sequence comprising part or allof RASSF1A, or other tumor suppressor genes or other genes whichdemonstrate aberrant DNA methylation patterns in cancer.
 5. The methodaccording to claim 4, wherein the DNA sequence is selected from thepromoter, exon 1, or fragments thereof of RASSF1A.
 6. The methodaccording to claim 5, wherein the DNA sequence is residues 1142 to 1269of SEQ ID NO:
 1. 7. The method according to claim 6, wherein: (a) theDNA sequence is amplified using a primer comprising the sequence shownin SEQ ID NO 2 and a primer comprising the sequence shown in SEQ ID NO:3; and/or (b) the DNA sequence is detected using a detectably-labelledprobe comprising the sequence shown in SEQ ID NO:
 4. 8. The methodaccording to claim 1, wherein following the enzymatic treatment step(b), each target DNA molecule of the sequence of interest in the sampleis cut at at least one methylation-sensitive restriction enzyme site bythe methylation-sensitive restriction enzyme(s) when present in theunmethylated state.
 9. The method according to claim 1, wherein in step(b) the sample is treated with an excess of the enzyme(s), and/orwherein the incubation time is extended.
 10. The method according toclaim 1, wherein the level of the DNA sequence quantified after theenzymatic treatment is used to: (a) diagnose cancer in the individual(b) predict the susceptibility to cancer of the individual; (c) assessthe stage of the cancer in the individual; (d) predict the likelihood ofoverall survival for the individual; (e) predict the likelihood ofrecurrence for the individual; or (f) assess the treatment effectivenessfor the individual.
 11. The method of claim 10, wherein the trend of thelevel of the DNA sequence after the enzymatic treatment over the timecourse of treatment, monitoring or post-treatment is quantified.
 12. Themethod according to claim 11, wherein the DNA sequence comprises part orall of RASSF1A and wherein the method is used in the diagnosis,prognosis or monitoring of hepatocellular carcinoma or nasopharyngealcarcinoma.
 13. The method according to claim 12, wherein the individualcarrier.
 14. The method according to claim 13, wherein the individual isa hepatitis B carrier or a hepatitis C carrier.
 15. The method accordingto claim 1, wherein the method further comprises quantifying ordetecting a control DNA sequence in the DNA sample that has beendigested with one or more methylation-sensitive restriction enzyme(s)wherein said control sequence does not demonstrate aberrant DNAmethylation patterns in cancer.
 16. The method according to claim 15,wherein at least two methylation-sensitive restriction enzymerecognition sites are present in the control sequence.
 17. The methodaccording to claim 16, wherein the same number of methylation-sensitiverestriction enzyme recognition sites are present in the control sequenceand the target sequence.
 18. The method according to claim 15, whereinthe control sequence is β-actin.
 19. The method according to claim 18,wherein a target sequence in the β-actin is amplified using a primercomprising the sequence shown in SEQ ID NO: 5 and a primer comprisingthe sequence shown in SEQ ID NO:
 6. 20. The method according to claim19, wherein the target sequence is detected using a detectably-labelledprobe comprising the sequence shown in SEQ ID NO:
 7. 21. The methodaccording to claim 15, wherein the method is used to assess the extentor to confirm the completeness of enzyme digestion.